Cholesterol is the major steroid constituent of animal tissue, and is a normal component of plasma and essentially all cell membranes. Cholesterol is a hydrophobic hydrocarbon compound with a reactive hydroxyl group. It is a 3-hydroxy sterol (having a perhydro-1,2-cyclopentenophenanthrene ring system skeleton) with an aliphatic side chain at position 17. In theory, it plays a major role in regulating the fluidity and permeability of the cell membrane, by forming an intercalated structure among the membrane phospholipids.
Cell membranes can experience transitions between a fluid-like consistency and a gel-like consistency, a phenomenon that is highly temperature sensitive. As a result, a membrane that is relatively fluid at 37.degree. C. can rapidly gel at temperatures only a few degrees lower.
In theory, cholesterol can prevent a temperature induced fluid to gel transition (a reduction in fluidity) by preventing the fatty acyl chains of the membrane lipids from binding with each other. This is achieved through its orientation within the membrane structure: the polar hydroxyl group of cholesterol is grounded toward the aqueous surface of the membrane and the polar heads of the membrane phospholipids, while the steroid ring is interposed between the fatty acyl side chains and keeps them apart. Darnell, et al., Molecular Cell Biology, Scientific American Books (New York: 1986), p. 576-577. Cholesterol can be obtained from the diet or synthesized in the liver. It can also be manufactured within the cell, if needed, but in practice most cells receive their cholesterol externally. Cholesterol synthesis occurs according to an enzyme-mediated biosynthetic pathway, in which the rate limiting enzyme is believed to be hydroxymethylglutaryl-Co-A reductase ("HMG CoA reductase"). See, for example, Javitt, U.S. Pat. No. 4,427,668. This enzyme catalyzes the formation of mevalonic acid, a cholesterol precursor, from hydroxymethyl glutaric acid. In theory, cholesterol production can be regulated by interfering with HMG CoA reductase. Goodman & Gilman, The Pharmacological Basis of Therapeutics, 7th ed., MacMillan (New York: 1985), pp. 841-843; Kandustsch et al., Science, 201, 498 (1978).
Cholesterol is insoluble in body fluids, and therefore must be transported through the bloodstream by a carrier. Perhaps the most clinically significant carrier is low-density lipoprotein (LDL), which can be envisioned as an apolar core of cholesterol (as a fatty acid ester) encapsulated within a spherical phospholipid monolayer membrane. The LDL membrane is embedded with a specific hydrophobic protein. Serum LDL particles have a diameter of from 20 to 25 nm, and are the major source of cholesterol for most cells. Darnell, supra., p. 648-650.
Accordingly, most cells manufacture specific receptors that will bind LDL to the cell membrane. Cholesterol can be internalized by cells bearing appropriate receptors, such as fibroblast cells, by receptor-mediated endocytocis. This is a process in which LDL forms a ligand-receptor complex by binding to the LDL receptor on the exterior of the cell membrane. Through a complex mechanism, LDL is transported across the membrane as a ligand-receptor vesicle, the receptor is recycled (in budded vesicles) to the membrane surface, and LDL is transported to the lysosomes (still in vesicles), where it is degraded to release cholesterol for use in the cell membrane. Id. Thus, it is believed that cholesterol accumulation (cellular uptake) and synthesis can be regulated by interfering with LDL binding.
The excessive accumulation of cholesterol has been implicated as the prime causative factor in a number of diseases. In particular, elevated concentrations of cholesterol can cause and/or hasten atherosclerosis, which is characterized by an abnormal hardening and thickening of the arterial walls due to the accumulation and deposition of fatty materials, including cholesterol. This, in turn, can lead to thrombosis and infarction. Accordingly, there is a need for means to therapeutically regulate cholesterol levels, especially when those levels become abnormally high. See, for example, Goodman & Gilman, pp. 827, 832.
Representative of known drugs that are currently in use are mevinolin and cholestyramine, which cause the upregulation of LDL receptors in the liver. Mevinolin, which is an allosteric inhibitor of HMG CoA reductase, achieves this regulation effect by inhibiting cholesterol synthesis in the liver. This stimulates increased LDL receptor activity in the liver, causing hepatic uptake of cholesterol, which in turn reduces plasma cholesterol. Cholestyramine increases the need for bile acid synthesis from cholesterol in the liver, which in some individuals will result in upregulation of LDL receptors. Once again, this reduces plasma cholesterol. The primary effect of both compounds is therefore limited to the liver. The known compounds are not intended to and do not significantly effect cells outside the liver. A strategy targeted for the liver is effective using the known compounds because, unlike other cells, liver cells can uniquely metabolize (and remove) cholesterol to bile acids. Indeed, compounds such as mevinolin would have a detrimental effect on the accumulation of tissue cholesterol if targeted on cells outside the liver, since the compound would upregulate the LDL receptors and would cause rather than reduce cholesterol accumulation.
Another known compound is 26-hydroxycholesterol, which is the subject of commonly owned U.S. Pat. No. 4,427,668, ("the '668 patent") the disclosure of which is hereby incorporated by reference. See also, Javitt et al., J. Biol. Chem, 256, 24: 12644-12646 (1981); Brooks et al., Biochem Soc. Trans., 11: 700-701 (1983); Koopman et al., J.Chromatogr., 416: 1-13 (1987); Easterman and Javitt et al., J. Lipid Res., 24: 1304-1309 (1983); and Taylor et al., J. Biol. Chem., 259, 20: 12382-12387 (1984).
The '668 patent suggests that certain derivatives and analogs of 26-hydroxycholesterol might be worthy of investigation as regulators of cholesterol production. By "derivative" and "analog" the patent expressly contemplates fatty acid mono and di-esters, sulfates, carbonates, glucuronides and ether or fluoro substitutions of 26-hydroxycholesterol. The patent does not disclose that any particular derivative or analog in fact exhibits biological activity as an inhibitor of cholesterol, HMG CoA reductase or LDL binding. The patent merely suggests a possible avenue for research. Moreover, there is no suggestion that any cholesterol reducing compound is a downregulator of LDL receptor binding or can be targeted specifically for non-hepatic (e.g., fibroblast) cells.
It should also be noted that there is no reliable correlation between liver or plasma cholesterol levels and the amount of HMG CoA reductase and LDL receptor binding. For example, the known drug mevinolin has been observed to inhibit hepatic cholesterol synthesis even though it increases the amount of HMG CoA reductase. The enzyme is less active in the presence of the drug, which is believed to modify mevinolin's three-dimensional conformation. From cell culture studies it is known that this drug (and others) also upregulates LDL receptors in fibroblasts and other non-hepatic cells. In contrast, 26-aminocholesterol and the derivatives and analogs herein inhibit HMG CoA reductase activity in fibroblasts and reduces LDL binding, thereby decreasing the rate at which tissue cells can produce and accumulate cholesterol. See also, Javitt et al., "Cholesterol metabolism: use of D.sub.2 O for determination of synthesis rate in cell culture," J.Lipid Res., 26: 950-954 (1985); Lorenzo et al., "Regulation of low density lipoprotein metabolism by 26-hydroxycholesterol in human fibroblasts," FEBS Letters, 218: 1, 77-80 (1987).
The drugs currently in use for the inhibition of cholesterol synthesis have an impact primarily on the liver, and are believed to function by causing upregulation of the LDL receptors of the liver (to increase liver uptake of cholesterol and decrease serum levels) or by inhibiting HMG CoA reductase, so that cholesterol production is reduced. The present compounds produce observably different results, in that LDL receptor binding of tissue cells is downregulated, while hepatic cells are not affected.